Activation of the mitochondrial permeability transition pore (PTP) clearly plays a key role in some of the most wide-spread and therapeutically challenging human diseases. Our studies have established that the PTP operates in two modes 1) transiently, whereby the PTP acts as a mitochondrial Ca2+ release channel or 2) persistently, which ultimately results in cell death and disease. Although well characterized on a functional level, we know remarkably little about the molecules that form the PTP or how it is regulated. We urgently need answers to basic questions concerning what proteins actually form the PTP channel and what modulates the opening of the PTP in vivo. As a result, our goal is to identify the molecules that contribute to the structure and regulation of the PTP in both normal and disease states. This information is critical if we are to be able to effectively identify and/or deign valuable therapeutics targeting the transition of the PTP from normal to pathological. Here, we will use biochemical and genetic tools to identify structural components of the PTP and how PTP activity can be dynamically regulated in vivo. The specific objectives of this application are based in the synergy possible through the unique combination of novel approaches available in the Forte and Bernardi laboratories. The specific objectives of this application are: Aim 1 - Test role of OMM proteins in the regulation of PTP activity: While the PTP is primarily an IMM event, it has long been appreciated that proteins in the OMM should prominently regulate PTP activity. We will initially focus on a specific OMM protein, Tspo, whose role in PTP regulation has been strongly suggested. Our studies here will allow us to gain a deeper understanding of how cytosolic elements can impact PTP activity. Aim 2 - Identify key structural components of the PTP: Despite our increasing appreciation of its fundamental role in normal and pathological cellular responses, the molecules that form the PTP have remained a mystery. Here, we will use information in available mitochondrial proteomes to identify proteins forming the pore of the PTP. It is our expectation that the identification of any single component forming the PTP will supply us with the missing hook, providing the opportunity to identify additional components. Aim 3 - Mitochondrial p66ShcA and ROS activation of the PTP: It is clear that pore open-closed transitions can be regulated at many levels and, by extension; misregulation of these upstream pathways can lead to persistent, pathological activation of the PTP. The goal of this aim is to investigate the hypothesis that ROS generated through the action of p66ShcA (p66) functions upstream of the activation of the PTP in conditions of excess oxidative stress. We anticipate on the completion of this aim to have clear understanding of the role of one novel upstream activator of PTP activity. These studies will set the stage for future interrogation aimed at extending our understanding of mitochondria and PTP activity in physiological and pathological settings. Clearly, these outcomes will be fundamental to developing novel therapeutic strategies specifically targeting the pore in the many disease processes in which the PTP has been clearly implicated.